Fibrosis model on a chip

ABSTRACT

The presently disclosed subject matter provides a biomimetic organ model, and methods of its production and use. In one exemplary embodiment, the biomimetic organ model can be a multi-layer model including a at least two microchannels and at least one chamber slab with at least one membrane coated with cells disposed between at least one microchannel and the at least one chamber slab. In another exemplary embodiment, the biomimetic organ disease model can be a five-layer model including a first and second microchannel with a membrane-gel layer-membrane coated or encompassing cells disposed between the microchannels. In certain embodiments, at least one device can be coupled to the biomimetic organ model that delivers an agent to at least one microchannel.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 62/197,444, filed on Jul. 27, 2015, U.S. Provisional ApplicationSer. No. 62/348,036, filed on Jun. 9, 2016, and U.S. ProvisionalApplication Ser. No. 62/348,055, filed on Jun. 9, 2016, all of which areincorporated by reference herein in their entirety.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under Director's NewInnovator Award No. 1DP2HL127720-01 awarded by the National Institute ofHealth. The government has certain rights in the invention.

BACKGROUND

The structural, functional and environmental complexity of human organsposes certain technical challenges for in vitro investigation of organphysiology and pathology using traditional cell culture models. As aresult, research in this area has relied on expensive and time-consumingex vivo or in vivo animal studies that can often fail to modelbiological responses in humans. These drawbacks of existing models canlimit the understanding and the development of new therapeuticapproaches to diseases. Therefore, there is a need for a low-cost, humancell-based alternative to current disease models.

For example, organ fibrosis is a progressive, life-threatening medicalcondition characterized by excessive deposition of extracellular matrix(ECM) in the connective tissue, leading to impairment of normal organarchitecture and function. Despite the increasing prevalence of fibrosisin various fatal diseases, understanding of its development andprogression remains rudimentary due to the failure of existing models torecapitulate complex human-relevant fibrotic responses.

One approach to meeting these challenges is to leverage microengineeringtechnologies that provide unprecedented capabilities to control cellularmicroenvironment with high spatiotemporal precision and to presentliving cultured cells with external influences and biochemical signalsin a more physiologically relevant context. This has led to thedevelopment of microengineered biomimetic systems such as“organs-on-chips” that simulate complex organ-level physiology. However,there remains a need for additional physiologically relevant, humancell-based alternatives to model organ disease. However, there remains aneed for additional physiologically relevant, human cell-basedalternatives to model fibrosis.

SUMMARY

The presently disclosed subject matter provides a biomimetic fibrosismodel (e.g., lung, liver, kidney, heart, skin, penile, brain, softtissue, or joint) and methods of its use. The present disclosure alsoprovides for methods of fabricating the biomimetic fibrosis model. Incertain embodiments, the biomimetic fibrosis model contains most of themajor cellular constituents of the organ being modeled. The biomimeticfibrosis model can be constructed from any organ from whichepithelial/parenchymal, stromal, and vascular cells can be obtained andcultured.

In certain embodiments, the biomimetic fibrosis model can be amulti-layer model which can include a body, at least one membrane, atleast one gel layer adjacent to the at least one membrane, at least onelayer of cells, and a device coupled to the body that can deliver anagent to the cells. In certain embodiments, a multi-layer model allowsthe modeling of epithelial and/or parenchymal (e.g., lung epithelialcells, liver hepatocytes, etc . . . ) compartments in one microchannel,stromal (e.g., extracellular matrix and organ specific such as lung orliver derived, fibroblasts, pericytes, stromal cells etc . . . )compartments in a gel layer, and vascular endothelial (e.g., lung orliver endothelial cells) compartments in another microchannel. Incertain embodiments, the multi-layer model can include vasculaturewithin the gel layer. In certain embodiments, the stromal compartmentcan include three-dimensional networks of blood and/or lymphatic vesselsthat can be perfused with blood, drugs, culture media, cells, toxins,particulates, and other materials. In certain embodiments, the stromalcompartment can include microchannels that can be perfused with blood,drugs, culture media, cells, toxin, particulates, and other materials.In certain embodiments, the inner surfaces of the microchannels withinthe stromal compartment can be coated with vascular or lymphaticendothelial cells. In certain embodiments, at least one layer of cellscan further comprise macrophages, dendritic cells, and/or microbialcells. In certain embodiments, the stromal compartment can containresident immune cells such as macrophages and dendritic cells. Incertain embodiments, the cells are derived from healthy organs, tissuesand/or body fluids. In certain embodiments, the cells can be derivedfrom diseased organs, tissues and/or body fluids. In certainembodiments, the diseased organ can be chronically diseased. In certainembodiments, the cells can be stem cell-derived cells. In certainembodiments, the cells can be patient-derived disease cells and/orpatient-derived stem cells.

In certain embodiments, the multi-layer biomimetic fibrosis model can bea three-layer model which includes a body, membrane, at least one layerof cells, and a device coupled to the body that can deliver an agent tothe cells. In certain embodiments, the body can have a first and secondmicrochannel. In certain embodiments, the first microchannel can besituated above the second microchannel. In certain embodiments, themembrane can be disposed between a first and second microchannel. Themembrane can have a first and second side, wherein the first side facesa first microchannel and the second side faces a second microchannel.The layer of cells can be disposed on the first side of the membrane. Incertain embodiments, the three-layer model further comprises a gel layerattached to the second side of the membrane. In certain embodiments, thecells can be embedded in the gel layer.

In certain embodiments, the multi-layer biomimetic fibrosis model can bea five-layer model which includes a body, membrane, at least one layerof cells, a gel layer, and a device coupled to the body that can deliveran agent to the cells. In certain embodiments, the body can have atleast a first and at least a second microchannel. In certainembodiments, the first microchannel can be disposed above the secondmicrochannel. In certain embodiments, at least one membrane can beadjacent to at least one microchannel. In certain embodiments, the firstmembrane adjacent to a first microchannel can have a first and secondside, wherein the first side faces a first microchannel and the secondside faces the gel layer. In certain embodiments, the second membraneadjacent to a second microchannel can have a first and second side,wherein the first side faces the gel layer and the second side faces asecond microchannel. A layer of cells can be disposed on the first sideof the first membrane. A layer of cells can be disposed on the secondside of the second membrane. In certain embodiments, a layer of organepithelial and/or parenchymal cells can be attached to the first side ofa first membrane. In certain embodiments, a layer of vascular and/orlymphatic endothelial cells can be attached to the second side of asecond membrane. In certain embodiments, a layer of vascular and/orlymphatic endothelial cells can be attached to the first side of a firstmembrane. In certain embodiments, a layer of organ epithelial cells canbe attached to the second side of a second membrane. In certainembodiments, the cells can be embedded in the gel layer.

In certain embodiments, the multi-layer biomimetic fibrosis model can bea three-layer model which includes a body, at least one layer of cells,a gel layer, and a device coupled to the body that can deliver an agentto the cells. In certain embodiments, the body can have at least a firstand a second microchannel. In certain embodiments, the firstmicrochannel can be disposed above the gel layer. In certainembodiments, the second microchannel can be disposed under the gellayer. In certain embodiments, a layer of cells can be attached to theupper side of the gel layer. In certain embodiments, a layer of cellscan be attached to the lower side of the gel layer. In certainembodiments, the cells can be embedded in the gel layer.

In certain embodiments, the five-layer model further comprises a gellayer disposed within the chamber. In certain embodiments, the chambercan have a width of about 3 mm×about 6 mm×about 1 mm. In certainembodiments, the chamber can have a width larger than 3 mm×about 6mm×about 1 mm. In certain embodiments, the chamber can have a widthlarger than 3 mm×about 6 mm×about 1 mm. In certain embodiments, the gellayer can be between each membrane. In certain embodiments, themembrane-gel layer-membrane structure can be disposed between a firstand second microchannel.

In certain embodiments, the gel of the multi-layer model can compriseextracellular matrix proteins such as, but not limited to, collagen,fibronectin, laminin, elastin, hyaluaronic acid, and/or similarmaterials. In certain embodiments, the gel can comprise collagen. Incertain embodiments, tissue or cells can be embedded in the gel. Incertain embodiments, engineered particles can be embedded in the gel. Incertain embodiments, sensors can be embedded in the gel. In certainembodiments, actuators can be embedded in the gel. In certainembodiments, the gel layer allows the embedded cells to communicate withone another and/or the layer of cells on the membrane and/or the surfaceof the gel. In certain embodiments, the membrane layers adjacent to thegel layer dissolve allowing the layer of cells on the membrane todirectly interact with the cells embedded in the gel layer.

In certain embodiments, the microchannel replicates the dimensions ofthe functional units of the organ being modeled (e.g., lung, liver,kidney, heart, skin, penile, brain, soft tissue, or joint). In certainembodiments, the microchannel replicates the dimensions of the airwaysin the native human lung or liver. In certain embodiments, the firstmicrochannel has a width from about 0.1 mm to about 2 mm. In certainembodiments, the first microchannel has a height from about 0.1 mm toabout 2 mm. In certain embodiments, the first microchannel has a lengthfrom about 1000 μm to about 30 mm.

In certain embodiments, the membrane of the model can be a porousmaterial. In certain embodiments, the membrane can comprise polyesterthin clear fabric, polydimethylsiloxane, polymeric compounds, or naturalmembranes, wherein the natural membrane comprise collagen, laminin,fibronectin, vitronectin, fibrin, other extracellular matrix proteins,fibroin, or a combination thereof.

In certain embodiments, the gel layer of the model contains theinterstitial and/or connective tissue of the organ. In certainembodiments, the gel layer of the model contains muscular tissue of theorgan, In certain embodiments, the gel layer of the model containsosseous tissue of the organ. In certain embodiments, the gel layer ofthe model contains neural tissue of the organ. In certain embodiments,the gel layer of the model contains adipose tissue of the organ. Incertain embodiments, the gel layer can further comprise macrophages,dendritic cells, and/or microbial cells.

In certain embodiments, the first microchannel can have culture mediumflowing through the microchannel. In certain embodiments, the secondmicrochannel can serve as a reservoir for basal feeding. In certainembodiments, the second microchannel can have culture medium flowingthrough the microchannel. In certain embodiments, the secondmicrochannel can have cell media held within its reservoir. A device candeliver an agent to at least one microchannel. In certain embodiments,the device can deliver an agent to the first microchannel and/or secondmicrochannel. In certain embodiments, the device can deliver an agent toone of a first or second microchannels. In certain embodiments, thedevice can deliver the agent to a first microchannel.

In certain embodiments, the agents can be small molecules, hormones,proteins, or peptides. In certain embodiments, the agents can be cells,tissue, functional particles, drug delivery vehicles, miniaturizedsensors, or miniaturized actuators. In certain embodiments, the devicedelivers the agent to the first microchannel and/or second microchannel.In certain embodiments, the device can deliver the agent to theperfusable vascular and/or lymphatic vessels within the gel layer. Incertain embodiments, the device can deliver the agent to themicrochannels within the gel layer. In certain embodiments, the devicecan deliver the agent directly to the gel layer. In certain embodiments,the device delivering the agent can be an automated machine.

The presently disclosed subject matter further provides methods forproducing a biomimetic fibrosis model. In certain embodiments, themethod can include fabricating a body. In certain embodiments, the bodycan have first and second microchannel disposed therein. In certainembodiments, the method can include inserting a membrane between thefirst and second microchannels. In certain embodiments, the membrane canhave a first side and a second side. In certain embodiments, the methodcan include adhering a layer of cells to the first side of the membrane.In certain embodiments, the layer of cells comprise epithelial cells(e.g., pulmonary, hepatic, renal, etc . . . ). In certain embodiments,the method can include integrating macrophage cells among the epithelialcells. In certain embodiments, the method can include attaching a gellayer to the second side of the membrane.

In certain embodiments, the method can include fabricating a body. Incertain embodiments, the method of fabricating a body can includeinserting a first membrane between the first microchannel disposedwithin a first channel slab and the chamber disposed within a chamberslab. In certain embodiments, the method of fabricating a body caninclude inserting a second membrane between the second microchanneldisposed within a second channel slab and the chamber disposed within achamber slab. In certain embodiments, the method of fabricating a bodycan omit the first membrane and/or second membrane. The first and secondmembrane can have a first side and a second side. In certainembodiments, the method can include adhering a layer of cells to thefirst side of the first membrane and/or a layer of cells to the secondside of the second membrane. In certain embodiments, the layer of cellscomprise epithelial cells (e.g., pulmonary, hepatic, renal, etc . . . ).In certain embodiments, the layer of cells comprise vascular endothelial(e.g., lung or liver endothelial cells). In certain embodiments, themethod can include integrating macrophages, dendritic cells, and/ormicrobial cells among at least one of the layers. In certainembodiments, the method can include forming a gel layer within thechamber. In certain embodiments, the method can include attaching a gellayer to the chamber.

In certain embodiments, the method can include casting a gel. In certainembodiments, the gel can be composed of collagen. In certainembodiments, tissue or cells can be embedded in the gel.

In certain embodiments, the different layers of the biomimetic organmodel can be combined in a modular fashion. In certain embodiments, themethod can include chemically bonding the fabricated layers of thebiomimetic organ model. In certain embodiments, mechanical binding ofthe layers of the biomimetic organ model allows the layers of cells tobe separated and examined separately, for example, following treatmentwith the agent. In certain embodiments, mechanically bonding the layersincludes a clamp (e.g., a screw clamp). In certain embodiments, themethod can include bonding the fabricated layers of the biomimetic organmodel using adhesive materials (e.g., double sided tape, polymericresins, Velcro, etc.). In certain embodiments, the method can includebonding the fabricated layers of the biomimetic organ model usingnegative pressure (e.g., vacuum).

In certain embodiments, the method can include coupling at least onedevice to the body that delivers an agent to at least one microchannel.In certain embodiments, the method can include delivering an agent tothe first microchannel and/or the second microchannel. In certainembodiments, the method can include delivering an agent to one of thefirst or second microchannels. In certain embodiments, the method caninclude delivering a culture medium through the first and/or secondmicrochannel. In certain embodiments, the method can include deliveringa culture medium through the first and/or second microchannel and thenexchanging the flow of medium to the flow of air (with or without theagent) through the first microchannel. In certain embodiments, themethod can include delivering a culture medium through blood orlymphatic vessels formed within the gel layer. In certain embodiments,the method can include delivering an agent to blood and/or lymphaticvessels in the gel layer. In certain embodiments, the method can includedelivering a culture medium through microchannels in the gel layer. Incertain embodiments, the method can include delivering an agent tomicrochannels in the gel layer.

In accordance with certain embodiments of the disclosed subject matter,a method of testing the effects of a toxic agent on the layer of cells.In certain embodiments, the method can include placing an agent ofinterest in one of the first or second microchannels. In certainembodiments, the method can include placing an agent of interest inblood and/or lymphatic vessels and/or microchannels embedded in the gellayer. In certain embodiments, the method can include simulatingphysiological conditions. In certain embodiments, the method can includemeasuring pathological responses to the agent. In certain embodiments,the method can include measuring tissue hardening or softening inresponse to the agent.

In certain embodiments, the platform can model organ injury (e.g.,fibrosis). In certain embodiments, the biomimetic organ model can be amodel of organ fibrosis. In certain embodiments, an agent can induce orinhibit fibrosis in the biomimetic fibrotic organ model. In certainembodiments, the biomimetic organ model can be a model of inflammatorydiseases. For example, the presence of macrophages allows for modelinginflammation.

The presently disclosed subject matter further provides methods of usingthe disclosed biomimetic organ model. In certain embodiments, thebiomimetic organ model can be used for identifying pharmaceuticalcompositions that can treat or prevent organ disease (e.g., fibrosis).In certain embodiments, the biomimetic organ model can be used foridentifying agents harmful to the organ.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a microengineered biomimetic multi-layer modelsubsections (100) according to certain embodiments.

FIG. 2A-B depicts (FIG. 2A) a microengineered biomimetic five-layermodel subsections (200) according to certain embodiments and (FIG. 2B) apackaged microengineered biomimetic five-layer model according to othercertain embodiments.

FIG. 3 depicts a schematic of an exemplary approach to themicroengineered biomimetic five-layer model cell layer construction.

FIG. 4 depicts a schematic representation of an exemplary methodaccording to the disclosed subject matter.

FIG. 5 depicts a schematic representation of an exemplary methodaccording to the disclosed subject matter.

FIG. 6 depicts a schematic representation of an exemplary methodaccording to the disclosed subject matter.

FIG. 7 depicts a schematic representation of an exemplary methodaccording to the disclosed subject matter.

FIG. 8 depicts the use of a gel immobilization technique in connectionwith sonic hedgehog-driven (SHH) fibrosis, including sonic hedgehog, apro-fibrotic signaling protein.

FIG. 9 depicts an exemplary clamp apparatus for mechanically bonding thedifferent layers of the biomimetic organ model together.

FIG. 10 depicts the cellular physiology of the stromal cells after 5days in culture. The arrows denote dead cells.

FIG. 11 depicts one embodiment of the cellular physiology of thecell-lined fluidic channels with the gel layer of the 5-layer model.

FIG. 12 depicts the effect of serum concentrations on cell viability anddensity.

FIG. 13 depicts fibroblast proliferation induced by varying the serumconcentration and culturing for 12 days or 16 days via staining offibronectin (FN) and smooth muscle actin (SMA).

FIG. 14 depicts fibroblast proliferation induced by varying the serumconcentration and culturing for 12, 16, or 28 days via staining offibronectin (FN) and smooth muscle actin (SMA).

FIG. 15 depicts detachment of the gel layer from the chamber induced byvarying the serum concentration and culturing for 16 days.

FIG. 16 depicts distinct stromal cell subsets and emergent fibrotic focifollowing culturing the gel layer in 0.2% serum for 16 days.

FIG. 17 depicts live/dead staining after long periods of culture. Thearrows denote the few dead cells.

FIG. 18 depicts the presence of Gli-1 in the stromal layer of thefive-layer model.

FIG. 19 depicts the use of a gel immobilization technique to study sonichedgehog-driven (SHH) fibrosis including sonic hedgehog, a pro-fibroticsignaling protein.

FIG. 20 depicts SRC kinase inhibition induced reduction in serum-inducefibrosis.

FIG. 21 depicts retinoic acid induced reduction in serum-inducefibrosis.

FIG. 22 depicts the presence of CD11b and CD206 in the stromal layer ofthe five-layer model.

FIG. 23 depicts the effect of M2

microenvironment promotion of fibrosis.

FIG. 24 depicts the presence of Gli-1 in the stromal layer of thefive-layer model in M2 conditioned media.

FIG. 25 depicts fibroblast proliferation in a five-layer liver model.

DETAILED DESCRIPTION

The present disclosure provides a microengineering approach to emulatingand probing organ disease processes in a tissue-engineeredmicroenvironment that recapitulates the complexity of each organ (e.g.,lung, liver, kidney, heart, penile, uterine, placental, eye, brain,intestine, skin, joints, testis, cervix, ovary, ear, nose, oral cavity,or bone). The disclosed biomimetic organ models can integrate epithelialcells, stromal tissue, vascular components, muscular tissue, neuraltissue, and/or immune cells. The incorporation of perfused vascularchannels can introduce the circulating immune cell component which canmodel the recruitment of immune cells under inflammatory conditions.

In certain embodiments, the biomimetic organ model disclosed herein canprovide opportunities to develop specialized human disease models thatcan use patient-derived cells to simulate complex human-specific diseaseprocesses for a variety of biomedical, pharmaceutical, toxicological,and environmental applications. For example, in certain embodiments, thebiomimetic organ model disclosed herein can be used to study organpathologies as well as other pathophysiologic processes that can occurin the organ being modeled. In certain embodiments, the biomimetic organmodel can be used for identifying pharmaceutical compositions that cantreat or prevent organ disease. In certain embodiments, the biomimeticorgan model can be used to identify molecular targets and signalingpathways that can be modulated pharmacologically to treat, delay, orprevent disease. Additionally, in certain embodiments, the biomimeticorgan model can be used as a screening tool to evaluate the safety andtoxicity of environmental exposures (e.g., chemicals, toxins), consumerproducts, biomedical devices, and drugs, and the transfer of chemicalsbetween compartments (e.g., tissues) of the organ being modeled. Incertain embodiments, a multi-organ model comprising multiple organmodels connected in series can be used to evaluate the safety andtoxicity of environmental exposures (e.g., chemicals, toxins), consumerproducts, biomedical devices, and drugs, and the transfer of chemicalsbetween the organ and surrounding tissue. In certain embodiments, thebiomimetic organ model can be used for identifying agents harmful to theorgan.

The present disclosure also provides a microengineering approach toemulating and probing organ disease processes in a tissue-engineeredmicroenvironment that recapitulates the complexity of the organ. Incertain embodiments, the platform can model organ fibrosis. In certainembodiments, an agent can induce or inhibit fibrosis in the biomimeticfibrotic organ model. In certain embodiments, the biomimetic organ modelcan be a model of inflammatory diseases. For example, the presence ofmacrophages allows for modeling inflammation. In certain embodiments,both resident and circulating immune cells can be integrated into one ormore of tissue compartments and/or cell layers.

In certain embodiments, the biomimetic organ model can be a model fororgan fibrosis. In certain embodiments, the fibrosis model entailsmeasuring fibroblast proliferation, fibroblast ECM production, and/orstiffening of the gel containing the cells and/or tissue, among otherspecific cellular-level outputs such as the expression ofdisease-relevant genes and proteins. The model for organ fibrosis allowsfor the study of changes in a stromal tissue corresponding to thedevelopment of fibrosis in the same way that a piece of solid organ froman animal or human can be analyzed, with the added benefit of modularitythat makes separation of tissue layers routinely achievable, a processwhich requires surgical expertise and dissection microscopy to performusing organs harvested from animals. For example, each tissue layer inthe model can be separately fixed, stained and examined by microscopy,or subjected to lysis buffers for the purpose of isolating proteins ornucleic acid to perform biochemical and/or molecular biologicalanalyses. In another example, the model tissue can be isolated from thebiomimetic organ model and processed for analysis of its mechanicalproperties such as stiffness, viscoelasticity, and ECM architecture.

Biomimetic Multi-Layer Fibrosis Model

The presently disclosed subject matter provides a biomimetic multi-layerorgan model. The term “layer” includes microchannel layers, gel layers,and membranes. The biomimetic multi-layer organ model can be used tomodel organs such as, but not limited to, lung, liver, kidney, heart,vagina, cervix, skin, penile, brain, soft tissue, or joint. In certainembodiments, the biomimetic organ model contains the number of layersneeded to model the appropriate number of tissue types. In certainembodiments, the biomimetic organ model contains at least 3, at least 4,at least 5, at least 6, at least 7, at least 8, at least 9, or at least10 layers. In certain embodiments, the biomimetic organ model contains3, 4, 5, 6, 7, 8, 9, or 10 layers. In certain embodiments, thebiomimetic organ model contains 3 layers. In certain embodiments, thebiomimetic organ model contains 4 layers. In certain embodiments, thebiomimetic organ model contains 5 layers. In certain embodiments, thebiomimetic multi-layer organ model includes one or more feeding channelsthat are not seeded with cells. In certain embodiments, the body furtherincludes one or more microfabricated openings or ports, providing accessfor inoculation and harvest of cells and agents from any givencompartment. The presently disclosed subject matter provides abiomimetic multi-layer fibrosis model. For the purpose of illustrationand not limitation, FIG. 1 provides an exemplary biomimetic four-layerfibrosis model 100. In certain embodiments, the biomimetic fibrosismodel can include a first channel slab 113, a second channel slab 114,and a membrane 120. In certain embodiments, the first channel slab 113and a second channel slab 114 can include a first microchannel 111 andsecond microchannel 112, respectively, disposed thereon. In certainembodiments, the biomimetic fibrosis model can include a first channelslab 113, a second channel slab 114, a membrane 120, and a gel layerattached to the second side of the membrane 122. A lower reservoir slabcan encase the gel. In short term experiments (8 days vs. up to 4 weeks)such as the one pictured in the disclosed embodiment, the gel may not bebound on all sides, with stromal ECM production in response to smokeexposure via the epithelial-seeded microchannel being the primaryoutput.

For the purpose of illustration and not limitation, FIG. 2 (A and B)provides an exemplary biomimetic five-layer fibrosis model 200. Incertain embodiments, the first channel slab 210 and a second channelslab 220 can include a first microchannel 211 and second microchannel221, respectively, disposed thereon. In certain embodiments, the chamberslab 230 can include a chamber 231, disposed thereon. In certainembodiments, the biomimetic fibrosis model can include a first channelslab 210, a second channel slab 220, a chamber slab 230, a firstmembrane 240, a second membrane 250, and a gel layer 260.

Channel Slabs

For the purpose of illustration and not limitation, the first channelslab and the second channel slab can include at least one channel ineach channel slab. In certain embodiments, the first and second channelslabs can each include additional microchannels (e.g., two, three, four,or more, total channels) disposed thereon. In certain embodiments, forevery channel in the first channel slab there is a channel in the secondchannel slab in the same location.

In certain embodiments, each microchannel will have at least onemembrane disposed therebetween (e.g., between the first channel slab 113and the second channel slab 114). In certain embodiments, eachmicrochannel will have at least one membrane (e.g., first membrane andoptionally second membrane) and a gel layer disposed therebetween (e.g.,between the first channel slab 210 and the second channel slab 220).

In certain embodiments, at least one membrane can dissolve, in whichcase the cells grown on the membrane would directly contact the gellayer. In certain embodiments, the first membrane and second membraneare absent (i.e., cells can be cultured directly on the gel layerwithout the intervening membranes).

In certain embodiments, the size of the microchannels can replicate thedimensions of the native human organ being modeled (e.g., lung, liver,kidney, heart, skin, penile, brain, soft tissue, or joint). For example,the size of the microchannels can replicate the dimensions of theairways in the native human lung, liver, or skin.

In certain embodiments, the microchannel can be as high as it is wideand/or as it is long. In certain embodiments, the microchannel theheight, width, and/or length are different. In certain embodiments, theheight and/or width can change along the length of the biomimetic organmodel.

In certain embodiments, the height, width, or length of themicrochannels can separately be from about 0.01 nm to about 1 cm. Incertain embodiments, the height, width, or length of the microchannelscan separately be from about 0.02 nm to about 8 mm, about 0.04 nm toabout 6 mm, about 0.06 nm to about 4 mm, about 0.08 nm to about 2 mm,about 0.1 nm to about 1 mm, about 0.2 nm to about 800 μm, about 0.4 nmto about 600 μm, about 0.6 nm to about 400 μm, about 0.8 nm to about 200μm, about 1 nm to about 100 μm, about 2 nm to about 80 μm, about 4 nm toabout 60 μm, about 6 nm to about 40 μm, about 8 nm to about 20 μm, about10 nm to about 10 μm, about 20 nm to about 8 μm, about 40 nm to about 6μm, about 60 nm to about 4 μm, about 80 nm to about 2 μm, about 100 nmto about 1 about 200 nm to about 0.8 μm, or about 400 nm to about 0.6μm.

In certain embodiments, the height, width, or length of themicrochannels can separately be from about 0.1 mm to about 2 mm wide. Incertain embodiments, the height, width, or length of the microchannelscan separately be from about 0.5 mm to about 1 mm. In certainembodiments, the height, width, or length of the microchannels canseparately be from about 0.5 mm to about 2 mm. In certain embodiments,the height, width, or length of the microchannels can separately be fromabout 1 mm to about 2 mm. In certain embodiments, the height, width, orlength of the microchannels can separately be from about 0.6 mm to about1.9 mm, from about 0.7 mm to about 1.8 mm, from about 0.8 mm to about1.7 mm, from about 0.9 mm to about 1.6 mm, from about 1 mm to about 1.5mm, or from about 1.2 mm to about 1.4 mm. In certain embodiments, theheight, width, or length of the microchannels can separately be at leastabout 0.5 mm, at least about 0.75 mm, at least about 1 mm, at leastabout 1.25 mm, at least about 1.5 mm, at least about 1.75 mm, or atleast about 2 mm. In certain embodiments, the height and/or width of themicrochannels can separately be about 10 mm and the length of themicrochannels can be about 10 cm. In certain embodiments, the height,width, or length of the microchannels can separately be about 100 μm toabout 500 μm. In certain embodiments, the height, width, or length ofthe microchannels can separately be about 100 μm to about 400 μm. Incertain embodiments, the height, width, or length of the microchannelscan separately be about 100 μm to about 300 μm. In certain embodiments,the height, width, or length of the microchannels can separately beabout 100 μm to about 200 μm. In certain embodiments, the height, width,or length of the microchannels can separately be about 110 μm to about190 μm, about 120 μm to about 180 μm, about 130 μm to about 170 μm, orabout 140 μm to about 160 μm.

In certain embodiments, the first and second microchannel can have thesame dimensions. In certain embodiments, the first and secondmicrochannel can have the different dimensions. In certain embodiments,the microchannels can be each separately about 0.01 nm to about 1 cmwide; about 0.01 nm to about 1 cm high and 0.01 nm to about 1 cm long.In certain embodiments, the microchannels can be each separately about0.1 mm to about 2 mm wide; about 0.1 mm to about 2 mm high; and about 1mm to about 10 mm long. In certain embodiments, the microchannel can beabout 1 mm wide×about 1 mm high. In certain embodiments, themicrochannel can be about 2 mm wide×about 2 mm high. In certainembodiments, the microchannel decrease size as the organ cavity does(e.g, decrease in size as airways in the lung do). For example, one endof the microchannel can be smaller than the other end.

In certain embodiments, for every microchannel in the first channel slabthere are the same number of microchannels in each additional channelslab. In certain embodiments, each channel slab can have a differentnumber of microchannels. In certain embodiments, each channel slab canhave microchannels in the same corresponding locations. In certainembodiments, each channel slab can have microchannels in differentlocations.

In certain embodiments, the microchannels can be rectangular,trapezoidal, round, oval, semi-circular or semi-elliptical shaped,semi-circular and square combination.

The number of channels and layouts of the channels, including shape anddimensions, can vary based on the design of the first channel slaband/or additional channel slabs. In certain embodiments, eachmicrochannel will have generally similar dimensions. In certainembodiments, the microchannels will have different dimensions.

In certain embodiments, the channel slabs can be made of any suitablematerial, for example and without limitation, glass, metal, alloy,plastic, wood, paper, and polymer. Suitable polymers include, but arenot limited to, PDMS. In certain embodiments, the channel slabs can bemade of any suitable material that can be molded by lithography, 3Dprinted or in any other way fabricated into the desired shape anddimensions. In certain embodiments, the each channel slab is made fromthe same material. In certain embodiments, the each channel slab is madefrom different materials.

Chamber Slab

The chamber slab includes a chamber disposed thereon. In certainembodiments, the size of each chamber can replicate the dimensions ofstromal tissues relevant to the native organ being modeled. For thepurpose of illustration and not limitation, the chamber slab 230 caninclude a chamber 231, disposed thereon (FIGS. 2A and B).

In certain embodiments, the chamber slab can include more than onechamber (e.g., two, three, four, or more) disposed thereon, with eachhaving a gel layer disposed therein. In certain embodiments, there is achamber for each microchannel in the first channel slab. In certainembodiments, there are a different number of chambers than microchannelsin the first channel slab. The number of chambers, including shape anddimensions, can vary based on the design of the chamber slab. In certainembodiments the chamber can be about 1 mm to about 5 mm wide.

In certain embodiments the chamber can be about 1.2 mm to about 4.8 mm,1.4 mm to about 4.6 mm, about 1.6 mm to about 4.4 mm, about 1.8 mm toabout 4.2 mm, about 2 mm to about 4 mm, about 2.2 mm to about 3.8 mm,about 2.4 mm to about 3.6 mm, about 2.6 mm to about 3.4 mm wide, orabout 2.8 mm to about 3.2 mm. In certain embodiments, the chamber has awidth of about 3 mm. In certain embodiments, the chamber has a width ofabout 15 mm.

In certain embodiments the chamber can be about 0.1 mm to about 2 mmhigh. In certain embodiments the chamber can be about 0.2 mm to about1.8 mm, about 0.4 mm to about 1.6 mm, about 0.6 mm to about 1.4 mm, orabout 0.8 mm to about 1.2 mm high. In certain embodiments, the chamberhas a height of about 1 mm. In certain embodiments, the chamber has aheight of about 3 mm.

In certain embodiments, the chamber can be about 2 mm to about 10 mmlong. In certain embodiments the chamber can be about 2.5 mm to about9.5 mm, about 3 mm to about 9 mm, about 3.5 mm to about 8.5 mm, about 4mm to about 8 mm, about 4.5 mm to about 7.5 mm, about 5 mm to about 7mm, or about 5.5 mm to about 6.5 mm long. In certain embodiments, thechamber has a length of about 6 mm. In certain embodiments, the chamberhas a length of about 10 cm.

For the purpose of illustration and not limitation, the chamber (e.g.,231) can be about 3 mm×about 6 mm×about 1 mm.

In certain embodiments, the chamber can be rectangular, trapezoidal,round, oval, semi-circular or semi-elliptical shaped, semi-circular andsquare combination. In certain embodiments, the geometry of the chamberchanges depending on the shape of at least one of the microchannels.

In certain embodiments, each chamber will have generally similardimensions. In certain embodiments, each chamber will have differentdimensions. In certain embodiments, each chamber slab can be made of anysuitable material, for example and without limitation, glass, metal,alloy, plastic, wood, paper, and polymer. In certain embodiments, eachchamber slab can be made of the same or different material.

In certain embodiments, the gel layer can contain materialsencapsulating biochemical payloads (e.g., drug). In certain embodiments,the gel layer can contain emulsions. In certain embodiments, the gellayer can contain magnetic materials. In certain embodiments, the gellayer can contain exothermic or endothermic materials. In certainembodiments, the gel layer can contain light emitting or absorbingmaterials. In certain embodiments, the gel layer can containmechanically actuatable materials. In certain embodiments, the gel layercan contain electrically actuatable materials.

In certain embodiments, the gel layer can contain perfusable hollowtubes. In certain embodiments, the hollow tubes can be perfused withculture media, blood, artificial blood, and other fluids. In certainembodiments, the hollow tubes in the gel layer can be coated withendothelial cells. In certain embodiments, the stromal compartment cancontain vascular and/or lymphatic tubes formed by self-assembly ofendothelial cells embedded in the matrix gel. In certain embodiments,the gel layer can contain hollow cavities.

In certain embodiments, the chamber slabs can be made of any suitablematerial, for example and without limitation, glass, metal, alloy,plastic, wood, paper, and polymer. Suitable polymers include, but arenot limited to, PDMS. In certain embodiments, the channel slabs can bemade of any suitable material that can be molded by lithography, 3Dprinted or in any other way fabricated into the desired shape anddimensions. In certain embodiments, the each chamber slab is made fromthe same material. In certain embodiments, each chamber slab is madefrom different materials.

In certain embodiments, the gel layer can be attached to the second sideof the membrane separating a microchannel from an underlying reservoirchannel, rather than being placed into the chamber.

Membranes

In certain embodiments, there is a membrane for each microchannel. Incertain embodiments, there is one membrane for multiple microchannels.In certain embodiments, a microchannel does not have a correspondingmembrane.

In certain embodiments, cells are grown on one side of the membrane. Incertain embodiments, cells are grown on both sides of the membrane.

In certain embodiments, the each membrane can each independently canhave about 0.4 μm to about 10 μm pores. In certain embodiments, thepores have a diameter from about 0.5 μm to about 9 μm, about 0.6 μm toabout 8 μm, about 0.7 μm to about 7 μm, about 0.8 μm to about 6 μm,about 0.9 μm to about 5 μm, about 1 μm to about 4 μm, about 1.5 μm toabout 3.5 μm, or about 2 μm to about 3 μm. In certain embodiments, thepores can be any suitable size. In certain embodiments, the pores canhave varying pore sizes.

In certain embodiments, the thickness of the membrane can be about 1 μmto about 1 mm. In certain embodiments, the thickness of the membrane canbe about 50 μm to about 950 μm, about 100 μm to about 900 μm, about 150μm to about 850 μm, about 200 μm to about 800 μm, about 250 μm to about750 μm, about 300 μm to about 700 μm, about 350 μm to about 650 μm,about 400 μm to about 600 μm, or about 450 μm to about 550 μm. Incertain embodiments, the thickness of the membrane can be about 100nanometers to about 5 μm. In certain embodiments, the thickness of themembrane can be about 200 nanometers to about 4 μm, about 300 nanometersto about 3 μm, about 400 nanometers to about 2 μm, about 500 nanometersto about 1 μm, about 600 nanometers to about 900 nanometers, or about700 nanometers to about 800 nanometers. In certain embodiments, thethickness of the membrane can be about 5 μm to about 100 μm. In certainembodiments, the thickness of the membrane can be about 10 μm to about90 μm, about 20 μm to about 80 μm, about 30 μm to about 70 μm, about 40micros to about 60 μm. In certain embodiments, the thickness of themembrane is at least about 5 μm, at least about 10 μm, at least about 20μm, at least about 30 μm, at least about 40 μm, at least about 50 μm, atleast about 60 μm, at least about 70 μm, at least about 80 μm, at leastabout 90 μm, or at least about 100 μm. In certain embodiments, themembrane can include porous portions and non-porous portions.

For the purpose of illustration and not limitation, FIG. 1 provides anexemplary biomimetic three-layer organ model 100 in which the membrane120 can be disposed between the first microchannel 111 and the secondmicrochannel 112. In certain embodiments, the membrane 120 can have afirst side 121 and a second side 122. In certain embodiments, the firstmicrochannel 111 and the second microchannel 112 can be in fluidcommunication through the membrane 120. In certain embodiments, thefirst microchannel 111 and the second microchannel 112 can be in fluidcommunication through the membrane 120 and a gel layer attached to thesecond side of the membrane 122. In certain embodiments, the membrane120 can dissolve, in which case the cells grown on the first side of themembrane 121 and the second side of the membrane 122 would directlycontact.

For the purpose of illustration and not limitation, FIG. 2 (A and B)provides an exemplary biomimetic five-layer organ model 200 in which thefirst membrane 240 can be disposed between the first microchannel 211and the chamber 231 such that the first microchannel 211 and the gellayer 260 can be in fluid communication through the first membrane 240.In certain embodiments, the first membrane 240 can have a first side 241and a second side 242. In certain embodiments, the second membrane 250can be disposed between the second microchannel 221 and the chamber 231such that the second microchannel 221 and the gel layer 260 can be influid communication through the second membrane 250. In certainembodiments, the second membrane 250 can have a first side 251 and asecond side 252. In certain embodiments, the first microchannel 211 andthe second microchannel 221 can be in fluid communication through thefirst membrane 240, the gel layer 260, and the second membrane 250. Incertain embodiments, the first microchannel 211 and the secondmicrochannel 221 can be in fluid communication through the firstmembrane 240 and the gel layer 260. In certain embodiments, the firstmembrane 240 and/or second membrane 250 can dissolve, in which case thecells grown on the first membrane 240 and/or second membrane 250 woulddirectly contact the gel layer 260. In certain embodiments, the firstmicrochannel 211 and the second microchannel 221 can be in fluidcommunication through the gel layer 260 (i.e., no membranes present).

Elimination of the membrane can create a direct contact between theepithelial and/or endothelial cells and the stromal gel layer.Elimination of the membranes can allow transmigration studies, in whichcells need to traverse the membrane barriers, to be conducted using theorgan models.

In certain embodiments, each membrane can each independently be a thinclear polyester fiber, a polyester membrane, a polytetrafluoroethylenemembrane, an elastomeric (e.g., poly(dimethylsiloxane) (PDMS),polyurethane) membrane, a paper membrane, an extracellular matrixmembrane, or a natural membrane derived from other biological sources.In certain embodiments, the natural membrane may include collagen,laminin, any combination thereof, and or any ECM material or naturalbiopolymer that can be acquired, including biopolymers such as chitosanand alginate. The selection of the pore sizes, materials and otherfeatures of the membrane can be varied based on the design of thebiomimetic organ model, the experimental goals, or other suitablemotivations. The dissolving membranes can include water solublematerials (e.g., alginate and Poly-vinyl alcohol (PVA)). In certainembodiments, the dissolving membranes can include non-crosslinked ECMmembranes (e.g., membranes derived from Matrigel) that only provide atransient barrier following rehydration.

Cell Layers

In certain embodiments, the biomimetic organ model contains most of themajor cellular constituents of the human organ. For example, but notlimited to, the layer of cells can be lung, liver, kidney, heart,penile, uterine, placental, eye, brain, intestine, skin, joints, testis,cervix, ovary, ear, nose, oral cavity, or bone derived cells.

In certain embodiments, the first or second cell layer can have anartificially induced pathology (e.g., fibrosis). In certain embodiments,the cell layers can be monolayers.

For the purpose of illustration and not limitation, FIG. 1 provides anexemplary biomimetic three-layer organ model 100 in which a first layerof cells 130 can be grown on the first side of the membrane 121 and/orthe second side of the membrane 122. In certain embodiments, a firstlayer of cells 130 can be grown on the first side of the first membrane130 and a second layer of cells 140 can be grown on the second side ofthe second membrane 122.

For the purpose of illustration and not limitation, FIG. 1 provides anexemplary biomimetic three-layer organ model 100 comprising a gel layerattached to the second side of the membrane 122, which can be comprisedof collagen matrix and/or organ specific fibroblasts/pericytes/stromalcells (e.g., lung or liver derived) as described further below. Incertain embodiments, tissue or cells can be embedded in the gel layer.In certain embodiments, the gel layer allows the embedded cells tocommunicate with the layer of cells on the membrane 120.

For the purpose of illustration and not limitation, FIG. 2 (A and B)provides an exemplary biomimetic five-layer organ model 200 in which afirst layer of cells can be grown on the first side of the firstmembrane 241 and/or the second side of the second membrane 252. Incertain embodiments, a first layer of cells can be grown on the firstside of the first membrane 241 and a second layer of cells can be grownon the second side of the second membrane 252.

For the purpose of illustration and not limitation, FIG. 2 (A and B)provides an exemplary biomimetic five-layer organ model 200 comprising agel layer 260, which can be comprised of collagen matrix and/or organspecific fibroblasts/pericytes/stromal cells (e.g., lung or liverderived) as described further below. In certain embodiments, tissue orcells can be embedded in the gel layer 260. In certain embodiments, thegel layer 260 allows the embedded cells to communicate with the layer ofcells on the first membrane 240 and/or the second membrane 250.

In certain embodiments, a layer of cells can be attached to one side orboth sides of a membrane. In certain embodiments, a layer of cells canbe attached to one side of a membrane that is opposite from the gellayer. In certain embodiments, a layer of cells can be attached to oneside of a membrane that is facing the gel layer.

In certain embodiments, a layer of organ epithelial cells can beattached to the first side of a first membrane. In certain embodiments,a layer of vascular endothelial cells can be attached to the second sideof a second membrane. In certain embodiments, a layer of vascularendothelial cells can be attached to the first side of a first membrane.In certain embodiments, a layer of organ epithelial cells can beattached to the second side of a second membrane.

In certain embodiments, the layer of cells can be epithelial cells(e.g., lung epithelial cells or liver hepatocytes). In certainembodiments, the epithelial cells can be from all compartments of theorgan. In certain embodiments, the epithelial cells are derived fromhuman or animal tissue. In certain embodiments, the epithelial cells canbe from healthy human or animal organ tissue. In certain embodiments,the epithelial cells can be from diseased human or animal organ tissue(e.g., fibrotic). In certain embodiments, the diseased organ can bechronically diseased. In certain embodiments, the layer of cells canfurther comprise macrophages, dendritic cells, and/or microbial cells.

In certain embodiments, the layer of cells can be endothelial cells(e.g., pulmonary or hepatic microvascular endothelial cells). In certainembodiments, the endothelial cells can be large vessel endothelialcells, arterial endohtelial cells, venous endothelial cells. In certainembodiments, the endothelial cells can be lymphatic endothelial cells.In certain embodiments, the endothelial cells are derived from human oranimal tissue. In certain embodiments, the endothelial cells can be fromhealthy human or animal organ tissue. In certain embodiments, theendothelial cells can be from diseased human or animal organ tissue(e.g., fibrotic). In certain embodiments, the diseased organ can bechronically diseased. In certain embodiments, the layer of cells canfurther comprise macrophages, dendritic cells, and/or microbial cells.

In certain embodiments, the multi-layer model further comprises a gellayer embedded with cells disposed within the chamber. In certainembodiments, the gel layer of the model contains the interstitial and/orconnective tissue or cells of the organ. For example, the gel of themodel can comprise extracellular matrix proteins such as, but notlimited to, collagen, fibronectin, laminin, elastin, hyaluaronic acid,and/or similar materials. In certain embodiments, the gel can comprisecollagen. Any collagen subtype can be used (e.g., collagen I, II, IV,and accessible collagen). Any extracellular matrix material can be used,including any organotypic mixture from tissues/organs. In certainembodiments, fibronectin, vitronectin, laminins, proteoglycans, tenascin(e.g., extracellular matrix glycoproteins) etc. can be used (e.g.,tenascin-C, —R, —X, and/or —W). In certain embodiments, the gel layercan further comprise macrophages, dendritic cells, and/or microbialcells.

In certain embodiments, tissue or cells can be embedded in the gel. Thegel layer allows the embedded cells to communicate with the layer ofcells on the membrane. In certain embodiments, the membrane layersadjacent to the gel layer dissolve allowing the layer of cells on themembrane to directly interact with the cells embedded in the gel.

In certain embodiments, the cells embedded in the gel layer can beconnective tissue or cells. In certain embodiments, the cells embeddedin the gel layer can be stromal cells. The cells embedded in the gellayer can be basal stromal cells, such as, but not limited to,fibroblasts and/or pericytes. In certain embodiments, the cells embeddedin the gel layer can be airway and/or vascular smooth muscle cells. Incertain embodiments, the cells embedded in the gel layer can be neurons,astrocytes, and microglia cells. In certain embodiments, the cellsembedded in the gel layer can be endothelial cells and fibroblasts ofany organ. In certain embodiments, the cells embedded in the gel layercan be osteocytes, osteoblasts, and osteoclasts. In certain embodiments,the cells embedded in the gel layer can be adipocytes and/or adiposetissue-derived stem cells. In certain embodiments, the cells embedded inthe gel layer can be sertoli cells and Leydig cells. In certainembodiments, the cells embedded in the gel can be cervical smooth musclecells. In certain embodiments, the cells embedded in the gel can beuterine smooth muscle cells. In certain embodiments, the cells embeddedin the gel layer can be dermal papilla cells. In certain embodiments,the cells embedded in the gel layer can be keratocytes, In certainembodiments, the cells embedded in the gel layer can be retinal cells.In certain embodiments, the cells embedded in the gel layer can bedermal papilla cells.

In certain embodiments, the layers of cells can further comprisemacrophages, dendritic cells, and/or microbial cells. In certainembodiments, the macrophages can be alveolar, interstitial,intravascular, airway macrophages and/or an immortalized cell line(e.g., THP-1). The macrophages can be harvested from tissue. Themacrophages can be generated. In certain embodiments, the macrophagescan be generated from blood (e.g., from peripheral blood monocytes). Incertain embodiments, the macrophages can be a primary component of thegel layer.

In certain embodiments, such as the embodiment of the four-layer model,the macrophage cells can be added to the epithelial cell layer forchannel co-culture at a ratio of about 1 macrophage to about 100epithelial cells. In certain embodiments, the macrophage cells can beadded to the epithelial cell layer at a ratio of about 1 macrophage toabout 50 epithelial cells. In certain embodiments, the macrophage cellscan be added to the epithelial cell layer at a ratio of about 1macrophage to about 95 epithelial cells, about 1 macrophage to about 90epithelial cells, about 1 macrophage to about 85 epithelial cells, about1 macrophage to about 80 epithelial cells, about 1 macrophage to about75 epithelial cells, about 1 macrophage to about 70 epithelial cells,about 1 macrophage to about 65 epithelial cells, about 1 macrophage toabout 60 epithelial cells, about 1 macrophage to about 55 epithelialcells, about 1 macrophage to about 50 epithelial cells, about 1macrophage to about 45 epithelial cells, about 1 macrophage to about 40epithelial cells, about 1 macrophage to about 35 epithelial cells, about1 macrophage to about 30 epithelial cells, about 1 macrophage to about25 epithelial cells, about 1 macrophage to about 20 epithelial cells,about 1 macrophage to about 18 epithelial cells, about 1 macrophage toabout 16 epithelial cells, about 1 macrophage to about 14 epithelialcells, about 1 macrophage to about 12 epithelial cells, about 1macrophage to about 10 epithelial cells, about 1 macrophage to about 8epithelial cells, about 1 macrophage to about 6 epithelial cells, orabout 1 macrophage to about 5 epithelial cells. In certain embodiments,similar ratios of macrophages to epithelial cells can be used forco-culture of macrophages with organ-specific fibroblasts in the gellayer.

In certain embodiments, the dendritic cells can be Langerhans cells,interstitial dendritic cells, interdigitating dendritic cells,follicular dendritic cells, and/or circulating dendritic cells.Dendritic cells and progenitors may be obtained from peripheral blood,bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltratingcells, lymph nodes, spleen, skin, umbilical cord blood or any othersuitable tissue or fluid.

In certain embodiments, the microbial cells can be bacteria, yeast,mold, lichens, algae, fungi, actinomycetes and/or protozoa.

In certain embodiments, circulating immune cells (e.g., neutrophils,eosinophils, basophils, lymphocytes, and/or monocytes) can be introducedinto the first and/or second microchannel (e.g., 111, 112, 211, 221) viaperfusion with the appropriate culture medium. Introduction of immunecells into the first and/or second microchannel can model therecruitment of immune cells under pathological conditions (e.g.,inflammatory).

Referring to FIG. 3 for the purpose of illustration and not limitation,there is provided a exemplary model of the order of the different celllayers of the five-layer model. For example, in FIG. 3, the epithelialcells are grown on the first side of the first membrane and theendothelial cells are grown on the second side of the second membranewhile the gel layer incorporates the stromal compartment. In certainembodiments, the exemplary model of FIG. 3 can be used for immune celltransmigration experiments. In certain embodiments, the cells areincorporated in a different order.

For the purpose of illustration and not limitation, the biomimetic organmodel can be a lung model containing a portion of the major cellularconstituents in the airway niches of the human lung. In certainembodiments, the layer of cells comprises airway epithelial cells. Incertain embodiments, the airway epithelial cells can comprise Type I andType II cells. In certain embodiments, the airway epithelial cells canbe from all compartments of the lung, including but not limited to,tracheal epithelial cells, bronchial epithelial cells, small airwayepithelial cells and/or alveolar epithelial cells, (e.g., Type I and IIcells). In certain embodiments, the second layer of cells can beendothelial cells including pulmonary microvascular endothelial cellssuch as large vessel endothelial cells, arterial endohtelial cells,venous endothelial cells all from lung. In certain embodiments, thesecond layer of cells can be lymphatic endothelial cells. In certainembodiments, the platform can model all the different segments/depths ofthe lung. In certain embodiments, the airway epithelial cells can befrom healthy human lung. In certain embodiments, the airway epithelialcells can be from a human diseased lung. In certain embodiments, thediseased lung can be chronically diseased. In certain embodiments, thelayer of cells can further comprise macrophages, dendritic cells, and/ormicrobial cells. In certain embodiments, the macrophages can bealveolar, interstitial, intravascular, airway macrophages, and/or animmortalized macrophage cell line (e.g., THP-1).

Cell Culture

In certain embodiments, the cells can be obtained from organ (e.g.,lung, liver, kidney, heart, penile, uterine, placental, eye, brain,intestine, skin, joints, testis, cervix, ovary, ear, nose, oral cavity,or bone) tissue. In certain embodiments, the cells can be obtained froma primary culture generated from the organ tissue. Standard techniquesof tissue harvesting and preparation can be used.

In certain embodiments, any of the cells can be derived from animmortalized cell line.

In certain embodiments, any of the cells can be stem cell-derived cells.

In certain embodiments, adhering the layer of cells to the first and/orsecond membrane can include standard approaches of extracellular matrixcoating of the membrane, for example, but not limited to the use offibronectin, prior to seeding of cells. In certain embodiments, adheringthe layer of cells to the first side of the membrane can includeformation of extracellular matrix hydrogel on the surface of themembrane, for example, but not limited to the use of collagen gel, priorto seeding of cells.

In certain embodiments, to seed the cells, a high density cellsuspension can be introduced to the channel and allowed to incubateunder static conditions to allow the cells to adhere. In certainembodiments, the cell suspension can be allowed to incubate for 2 to 4hours. In certain embodiments, the seeding method (e.g., incubationperiod of the cell suspension) can vary depending on the compartmentand/or cell type. In certain embodiments, after the period of attachmentflow can be initiated to allow the washing away of unattached cells andbeginning the perfused culture stage. In certain embodiments, some cellproliferation can occur to fill out the entire membrane surface. Incertain embodiments, cell proliferation is allowed to occur for 2-3 daysor longer.

In certain embodiments, the immune cells are obtained from peripheralblood and incorporated into the organ model. For example, peripheralblood monocytes can be obtained to generate the macrophage cells used inthe model. In certain embodiments, THP-1 cells are used. In certainembodiments, macrophages can be obtained from patient biopsies. Incertain embodiments, the macrophages can be obtained by organ lavages(e.g., but not limited to bronchoalveolar, renal, or vaginal lavages).

In certain embodiments, the macrophages can be introduced into thechannel after the epithelial layer has formed. For example, this can beaccomplished by pipetting them into the channel. As they differentiatethey can adhere to the epithelial cells and can migrate on theepithelial surface. In certain embodiments, inflammatory responses canbe assessed by testing the strength of macrophage adherence to theepithelium by washing the channel and assaying the number of macrophagesthat remain adherent to the epithelium.

In certain embodiments, the stromal cells are derived from a primarycell culture, established cell culture, or an immortalized cell culture.In certain embodiments, the stromal cells are obtained from a biopsiedtissue. In certain embodiments, the stromal cells can be derived frompatient-derived stem cells such as mesenchymal stem cells, or frompluripotent cells including healthy donor- or disease patient-derivedinduced pluripotent stem cells.

In certain embodiments, the gel layer contains nutrients to feed thecells. In certain embodiments, the cells in the gel layer obtainnutrients from culture medium from the microchannels or a reservoir. Incertain embodiments, the cells obtain nutrients from within the geland/or from culture medium from the microchannels or a reservoir.

Methods of Fabrication

In certain embodiments, the method can include fabricating a body, thebody having microchannel layers and at least one gel layer disposedthereon. The body, including the microchannels layers and gel layers,can be built by any methods known in the art, including, but not limitedto, those outlined in Huh et al., Nature Protocols 8:2135-2157 (2013).

In certain embodiments, the different layers of the body can bechemically bonded, i.e., oxygen plasma treatment of PDMS. Chemicallybonding can result in cell death; therefore, if the biomimetic organmodel is chemically bonded together, the cells can be added to thebiomimetic organ model after chemical bonding is complete.

In certain embodiments, the different layers of the body can bemechanically bonded. Mechanical bonding allows the different tissuecomponents to be cultured separately before interfacing them together,so as to engineer the various tissue layers in an isolated context andthen stacking them to form the organ (multi-tissue) configuration. Incertain embodiments, mechanically bonding the layers includes a clamp. Aclamp includes, but is not limited to a screw clamp, cam clamp, springclamp, binder clip, vice, C-clamp, adjustable hand screw clamp, springclamp, pipe clamp, bar clamp, parallel clamp, F style clamp, or athreaded rod with one or more fasteners. In certain embodiments, themethod can include bonding the fabricated layers of the biomimetic organmodel using adhesive materials. Adhesive materials includes, but are notlimited to, PDMS glue, double sided tape, hemming tape, removableadhesive fabric, rubber cement, adhesive polymers (e.g., polysulfones,polyethersulfones, polyimides, polyamide-imides, epoxy resins,polyarylene ether ketones such as, chloromethylated polyarylene etherketones, acryloylated polyarylene ether ketones, and mixtures thereof,preformed polyimides, polyetherimides, polystyrene, and the like andcholromethylated polyethersulfones and acryloylated polyethersulfones).In certain embodiments, the method can include bonding the fabricatedlayers of the biomimetic organ model using negative pressure (e.g.,vacuum).

In certain embodiments, the different layers of the biomimetic organfibrosis model can be combined in modular fashion according to a desiredtime sequence. In certain embodiments, the entire biomimetic organfibrosis model device does not need to assemble at first. For example,each of the layers can be cultured separately for any desired length oftime and then subsequently combined to form the complete model.

In certain embodiments, the method can include casting a gel in thechamber of the chamber slab. In certain embodiments, the method caninclude casting a gel to attach to a single membrane or multiplemembranes.

Gel casting can involve any standard method known to one of skill in theart. In certain embodiments, techniques are used to induce surfacemodification to promote collagen/ECM anchoring. In certain embodiments,the casting of a gel can include sulfo-sanpah treatment of the chamberslab material to promote collagen/ECM anchorage. In certain embodiments,the casting of a gel can include sulfo-sanpah treatment of the membranematerial and channel material to promote collagen/ECM anchorage. Forexample, the surface of the portion of the chamber slab in which the gellayer can be attached to can be treated with sulfo-sanpah and exposed toUV light (for example two times at 5 minutes each).

In certain embodiments, the gel layer can contain perfusable hollowtubes. In certain embodiments, the gel layer can contain hollowcavities. In certain embodiments, tubes and cavities can be formed byseveral techniques including needle withdrawal and vasculogenesis (e.g.,development of an interconnected hollow tubular network by vascularendothelial cells present in the gel layer). According to the needlewithdrawal technique, a gel can be formed around a thin needle that issubsequently removed to leave a channel in the gel. According to thevasculogenesis technique, vascular cells can be seeded in a channelinterfaced with the gel to grow into the gel, creating a network ofperfusable capillaries.

In certain embodiments, the gel is prepared with cells and pipetted ontothe chamber. The density of the cells can range from about 1×10⁴/ml ofgel solution cells to 1×10⁸/ml of gel solution, depending on theexperiment and the culture condition of the cells. One of ordinary skillwould understand the cell density and culture conditions required foreach particular gel layer depending on the goals of the experiment(e.g., lower density normal vs higher density fibrotic tissues).

In certain embodiments, the gel is prepared without cells and pipettedonto the chamber or onto a membrane. If the biomimetic organ model isalready bound together, the cells can be placed into one of the channelsand transmigrate to populate the empty gel.

In certain embodiments, the surface-anchored hydrogel extends the timebefore contraction of the gel layer away from the chamber occurs. Incertain embodiments, contraction of the surface-anchored hydrogel awayfrom the chamber occurs no earlier than about 2 days, about 3 days,about 4 days, about 5 days, about 6 days, about 7 days, about 8 days,about 9 days about 10 days, about 11 days, about 12 days, about 13 days,about 14 days, about 15 days, about 16 days, about 17 days, about 18days, about 19 days, or about 20 days. In certain embodiments,contraction of the surface-anchored hydrogel away from the chamberoccurs between about 5 and about 20 days, about 6 and about 19 days,about 7 and about 18 days, about 8 and about 17 days, about 9 and 16days, about 10 and about 15 days, about 11 and about 14 days, or about12 and about 13 days. In certain embodiments, contraction of thesurface-anchored hydrogel away from the chamber occurs no earlier thanabout 7 days, about 10 days, about 14 days, about 17 days, about 21days, about 24 days, about 28 days, about 31 days, about 35 days, about38 days, or about 42 days. In certain embodiments, contraction of thesurface-anchored hydrogel away from the chamber occurs no earlier thanabout 1 week or no earlier than about 2 weeks under fibrotic conditions.In certain embodiments, contraction of the surface-anchored hydrogelaway from the chamber occurs no earlier than about 3 weeks or no earlierthan about 4 weeks under normal (e.g., non-fibrotic) conditions.

Referring to FIG. 4 for the purpose of illustration and not limitation,there is provided an exemplary method for fabricating a biomimeticfive-layer organ model (400). In certain embodiments, the method caninclude fabricating a first channel slab, a second channel slab, and achamber slab (401), the first channel slab and second channel slabhaving a first and second microchannels disposed thereon and the chamberslab having a chamber disposed thereon. In certain embodiments, themethod can include casting a gel into the chamber of the chamber slab(402). In certain embodiments, the method can include inserting a firstmembrane between the first channel slab and the chamber slab and asecond membrane between the second channel slab and the chamber slab(403) such that the first and second microchannels can each be in fluidcommunication with the chamber through the membranes. In certainembodiments, temporary layers can be used to ensure the gel surfaces areflat and it can subsequently be contacted with a membrane-bound channel.The constituents of the gel are described above. In certain embodiments,the method can include adhering a first layer of cells (404) of a firstcell type disposed on a first side of the first membrane. In certainembodiments, the method can include adhering a second layer of cells(405) of a second cell type disposed on a second side of the secondmembrane. In certain embodiments, the first membrane and second membraneare absent (i.e., cells can be cultured directly on the gel layerwithout the intervening membranes) or the membranes dissolve.

Referring to FIG. 5 for the purpose of illustration and not limitation,there is provided an exemplary method for fabricating a biomimeticfive-layer organ model (500). In certain embodiments, the method caninclude fabricating a first channel slab and a second channel slab(501), wherein each portion has at least one separate microchanneldisposed thereon. In certain embodiments, the method can includefabricating a chamber slab (502), wherein the chamber slab has at leastone chamber disposed thereon. In certain embodiments, the cells areindividually cultured prior to the assembly of the biomimetic organmodel. In certain embodiments, the method can include casting a gellayer in the chamber (503). The constituents of the gel are describedabove. In certain embodiments, the method can include placing a firstmembrane to the first microchannel and a second membrane to the secondmicrochannel (504). In certain embodiments, the membranes can be fixedto the channel slabs via PDMS stamping or gluing. In certainembodiments, the PDMS glue can be uncured PDMS. In certain embodiments,the membranes can be held to the channel slabs using mechanical means asdiscussed above. In certain embodiments, the membrane can have a firstand second side. In certain embodiments, the slabs containing the firstand second microchannels and chamber can be mechanically bonded (505) asdiscussed above. The first side of the first membrane faces the firstmicrochannel and the second side of the second membrane faces the secondmicrochannel once the biomimetic organ model is assembled. In certainembodiments, the method can include (506) adhering a layer of cells of afirst cell type disposed on a first side of the first membrane. Incertain embodiments, the method can include (507) adhering a layer ofcells of a second cell type disposed on a second side of the secondmembrane.

Referring to FIG. 6 for the purpose of illustration and not limitation,there is provided an exemplary method for fabricating a biomimeticfive-layer organ model (600). In certain embodiments, the method caninclude fabricating a first channel slab and a second channel slab(601), wherein each portion has a separate microchannel disposedthereon. In certain embodiments, the method can include fabricating achamber slab (602), wherein the chamber slab has a chamber disposedthereon. In certain embodiments, the cells are individually culturedprior to the assembly of the biomimetic organ model (603). In certainembodiments, the method can include casting a gel layer in the chamber(604). The constituents of the gel are described above. In certainembodiments, the method can include placing a first membrane over thefirst microchannel and a second membrane over the second microchannel(605), wherein a layer of cells are adhered to at least one of themembranes. In certain embodiments, the slabs containing the first andsecond microchannels and chamber can be mechanically bonded (606) asdiscussed above. The first side of the first membrane faces the firstmicrochannel and the second side of the second membrane faces the secondmicrochannel once the biomimetic organ model is assembled.

In certain embodiments, a device can deliver culture medium to the firstand/or second microchannels. In certain embodiments, a device candeliver culture medium to one of the first or second microchannels. Incertain embodiments, a device can deliver culture medium to only thesecond microchannel. In certain embodiments, the device can pump culturemedium to the microchannel(s) through a port (e.g., FIG. 2 (e.g., 212and/or 222)) in the channel slabs, wherein the first opening of the port(e.g., 212 and/or 222) can be to the outside of the channel slabs andthe second opening of the port (e.g., 212 and/or 222) can be to therespective microchannel. In certain embodiments, channels from the topand bottom of the channel slabs that lead to the microchannels can beaccessed to provide agent and/or culture media to the microchannels. Incertain embodiments, access to both the top and sides of the channelscan be provided. In certain embodiments, the culture medium leaves themicrochannel through an exit port (e.g., 213 and/or 223). In certainembodiments, the device can pump culture medium out of themicrochannel(s) through an exit port (e.g., 213 and/or 223), wherein thefirst opening of the exit port (e.g., 213 and/or 223) opens to themicrochannel and the second opening of the exit port (e.g., 213 and/or223) can be to the outside of the channel slab. In certain embodimentsthe port (e.g., 212 and/or 222) or exit port (e.g., 213 and/or 223) onlyconnects to one microchannel. In certain embodiments, cell culture mediawith different constituents can be added to separate microchannels. Thecell culture media can be selected based on the type of cell grown onthe membrane facing the microchannel. In certain embodiments, thepumping system can draw/pull medium though the channels from areservoir. In certain embodiments, the pumping system can push mediumthough the channels from a reservoir. In certain embodiments, perfusioncan be achieved without pumps using gravity driven flow. In certainembodiments, a recirculatory flow loop can be used. In certainembodiments, the pumping system can deliver medium from a separate organmodule or modules.

In certain embodiments, the second microchannel can have cell media heldwithin its reservoir.

Agent Delivery

In certain embodiments, the agent can be delivered to the first and/orsecond microchannel. In certain embodiments, the device delivers anagent to the first and/or second microchannel. In certain embodiments,the device delivering the agent can be an automated machine. In certainembodiments, the agent can be more dilute the deeper it moves into themicrochannel. In certain embodiments, the agent can be delivered at theconcentration and intermittent schedule.

In certain embodiments, the device can deliver the agent to themicrochannel(s) through a port (e.g., FIG. 2 (e.g., 212 and/or 222)) inthe channel slab, wherein the first opening of the port (e.g., 212and/or 222) can be to the outside of the body and the second opening ofthe port(e.g., 212 and/or 222) can be to at least one microchannel. Incertain embodiments, a recirculatory flow loop can be used to deliverthe agent.

In certain embodiments, the agents can be small molecules, hormones,proteins, or peptides. In certain embodiments, the device delivers theagent to the first microchannel and/or second microchannel. In certainembodiments, the agent can be inflammatory mediators such as, but notlimited to, cytokines, growth factors, hormones (e.g., IFNg, LPS, IL-4,IL-13—molecules that promote differentiation of M1 or M2 macrophages).One of ordinary skill in the art would understand to select theappropriate agent(s) for the specific disease process being addressed inthe model.

In certain embodiments, the agent induces the disease state of the organmodel. For example, agents such as, but not limited to, peptides andgrowth factors known to be unregulated during specific diseaseconditions (e.g. transforming growth factor-beta, sonic hedgehog (SHH),connective tissue growth factor, and any other agent released by damagedtissue cells during organ injury that promotes fibrosis), organ injurycausing agents (e.g., smoking), chemical injury (e.g., Bleomycin), orinflammatory mediators such as, but not limited to, cytokines, growthfactors, hormones (e.g., IFNg, LPS, IL-4, IL-13, M2 macrophages) can beused to induce organ injury (e.g., fibrosis). In certain embodiments,the agent causes oxidative stress.

In certain embodiments, the active agent is used to inhibit or preventthe organ injury. For example, the agent can be a pharmaceuticalcompound such as PP2 (a specific inhibitor of integrin-associatedSRC-kinase signaling) or endogenous inhibitors of fibrosis in vivo suchas retinoic acid, Vitamin A, and ATRA. In another example, the agent canbe Prostaglandin PGE2. In another example, the agent can be integrininhibitors. In another example, the agent can be FAK inhibitors. Inanother example, the agent can be SRC-kinase inhibitors. In anotherexample, the agent can be any type of RTK inhibitor. In another example,the agent can be Rho-GTPase inhibitors. In certain embodiments, anyagent identified by one skilled in the art that is known to modulatedisease-related signaling pathways can be used for testing.

In certain embodiments, the biomimetic organ model optimizes air-liquidinterface culture. In certain embodiments, the first microchannel canhave air or gases flowing through the microchannel.

In certain embodiments, the substance of interest can be, for examplebut not limited to cigarette smoke, nicotine aerosol (e.g., e-cigarets,nicotine vapor), wood smoke, natural plant smoke, silica dust, acrylicdust, celestial dust, particulates, asbestos fibers, solvents, graindust, engineered nanomaterials, ultrafine particles, pathogenic species(e.g., viruses and bacteria), stem cells, dry powder drugs, aerosolizeddrugs, bird droppings, and animal droppings.

Referring to the lung for the purpose of illustration and notlimitation, the device delivers the agent to the first microchannel. Incertain embodiments, the method can include simulating physiologicalflow conditions. In certain embodiments, the method can includesimulating physiological breathing/inhalation conditions. In certainembodiments, the device delivering the agent can be an automatic agentdelivering machine. In certain embodiments, the agent can be deliveredto the first microchannel such that the distribution of the agent mimicsexposure conditions experience by cell linings in the human lung. Incertain embodiments, the agent can be more dilute the deeper it movesinto the first microchannel. The dilution can be finely tuned andmatched to predicted or measured values from smoke or particulates inhuman in vivo lungs.

In certain embodiments, for the airborne agent delivery (e.g. smoke),the airborne agent can be pulled through the biomimetic organ model. Incertain embodiments, there can be a mixing chamber in the apparatus(e.g., the smoke is generated and diluted/humified in a positivepressure flow process, it then fills an open mixing vessel) from whichthe airborne agent/air mixture can be pulled through the biomimeticorgan model at a set flow rate via syringe pump.

In certain embodiments, the culture medium is not delivered to thebiomimetic organ model while the agent is being delivered. In certainembodiments, the culture medium is delivered to one microchannel whilethe agent is delivered to the other microchannel. In certainembodiments, a recirculatory flow loop can be used to deliver the agent.

In certain embodiments, the device delivering the agent can be anautomatic smoking machine (e.g. FIG. 8). In certain embodiments, such adevice can be used for any volatile species and/or particulate inaddition to tobacco smoke. For example, but not limited to, theautomatic machine could be a benchtop-sized, automated smoking machineinterfaced with microfluidic devices (e.g., a Human Puff Profile modelcigarette smoking machine (CH Technologies)). In certain embodiments,the device can be configured to delivery virtually any chemical and/orparticulate species (e.g., electronic cigarette vapor, wood smoke,particulate toxicants, etc.) that can be nebulized/aerosolized such thatit can be mixed with humidified air in the same manner as tobacco smoke.In certain embodiments, the Weibel model can be used to estimateappropriate concentration ranges prior to delivery of the agent. Incertain embodiments, the agent can be intermittently delivered to modelthe frequency in which a human's lungs may experience the agent (e.g.,to model a heavy versus light smoker). In certain embodiments, theconcentration of the smoke mimics that of a lung exposed to secondhandsmoke.

In certain embodiments, agents can be delivered to serially connecteddevices lined with different types of epithelial cells (e.g., nasal,tracheal, bronchial, bronchiolar, and alveolar). In this respect, themodel is able to simulate delivery through the entire organ system.Referring to the lung for the purpose of illustration and notlimitation, the respiratory tract could be modeled by seriallyconnecting devices to administer an agent to a model of the nose, to amodel of the trachea, to a model of the large airways, to a model of thesmall airways, and finally to a model of the alveoli.

Methods of Use

Referring to FIG. 7 for the purpose of illustration and not limitation,an exemplary method of testing the regulation of fibrotic tissue (700)is provided. In certain embodiments, the method can include providing abiomimetic five-layer model (701) as disclosed herein, and can includedelivering an agent of interest in one of the first and/or secondmicrochannels (702). In certain embodiments, the method can includemeasuring a change in cellular physiology (703).

In certain embodiments, the first channel slab, second channel slab, andchamber slab can be separated and each cell compartment (e.g., layer ormatrix) can be examined separately. Once each slab is separated, each ofthe cell compartments can be separately fixed, stained, and/or examinedby microscopy. The cells can also be subjected to lysis buffers for thepurpose of isolating proteins or nucleic acids or performing biochemicaland molecular biological analyses.

In certain embodiments, the method can include measuring pathologicalresponses to the agent. In certain embodiments, the method can includemeasuring tissue hardening or softening in response to the agent. Incertain embodiments, the method can include measuring changes in theviscoelastic properties of the tissue in response to the agent. Incertain embodiments, the method can include measuring extracellularmatrix reorganization of the tissue in response to the agent. In certainembodiments, the method can include measuring inflammatory and otheradverse biological responses, for example, but not limited to,production of cytokines/chemokines and expression of adhesion molecules;production of enzymes (e.g., MMPs, TIMPs, LDH); activation of oxidativestress pathways; production of free radicals; activation ofpro-inflammatory pathways; endoplasmic reticulum (protein production)stress; production of extracellular matrix proteins; cell proliferation;gene expression changes; DNA damage; or cell apoptosis and necrosis(e.g., death).

In certain embodiments, the method can include measuring inflammatoryresponses, for example, but not limited to, production ofcytokines/chemokines & expression of adhesion molecules; activation ofoxidative stress pathways, endoplasmic reticulum (protein production)stress; DNA damage; or cell apoptosis (i.e., death).

In certain embodiments, the models of the instant disclosure, includingbut not limited to the five-layer model, can be used to examinefibrosis. In certain embodiments, the gel layer is attached to thechamber as described above. For example, as conditions becomeprofibrotic the gel will eventually detach from a corner or wall of thechamber due to contractile force generated by the cells within the gellayer. In certain embodiments, the collagen-anchored PDMS chamber canextend the time scale before contraction occurs. This can allow forgreater sensitivity of examining a treatment (e.g., drug treatment). Incertain embodiments, the culture conditions will affect how much thecells try to contract the gel, but it will not affect the method ofanchoring the gel. The culture conditions influence the behavior of thecells and will vary from conditions that promote low contractility topro-fibrotic conditions with high contractility. The latter would beachieved by a myriad of growth factors, increased serum concentration orusing an injury model (induced via a cellular response). In certainembodiments, the culture conditions could be varied but not impact thephysical anchoring. In certain embodiments, the platform can model afibrotic organ model. In certain embodiments, the biomimetic fibroticorgan model, an agent can induce or inhibit fibrosis.

In certain embodiments, the biomimetic organ models of the instantdisclosure can include additional elements, including additionalmembrane layers, for example but not limited to, integrated pumps,valves, bubble traps, oxygenators, gas-exchangers, in-linemicroanalytical functions, and other suitable elements. Such elementscan allow for additional control and experimentation using thebiomimetic organ model. In certain embodiments, the biomimetic organmodel can include features for automatically performing experiments onthe biomimetic organ model. For example, in some embodiments, the gellayer can incorporate magnetic materials, exothermic or endothermicmaterials, light emitting or absorbing materials, mechanicallyactuatable materials, electrically actuatable materials, or combinationsthereof. For example, in some embodiments, the biomimetic organ modelcan include automated valves, pumps, or fluid (e.g., liquid, gas, oremulsions) control mechanisms or automatic monitoring and testingmechanisms, such as sensors, detectors, or monitors. In certainembodiments, the biomimetic organ model can be configured to be coupledwith other sensors, detectors, or monitors not disclosed on thebiomimetic organ model. In certain embodiments, the biomimetic organmodel can be configured to be coupled with other bioanalytical platformsand methodologies (e.g., gel electrophoresis, capillary electrophoresis,western blotting, ELISA, mass-spectrometry) not disclosed on thebiomimetic organ model. In certain embodiments, the biomimetic organmodel can include a cleaning reservoir coupled to the channels forcleaning or sterilizing the channels. In certain embodiments, thebiomimetic organ model can be modular in construction, thereby allowingvarious elements to be attached or unattached as necessary duringvarious cleaning, experimenting, and imaging processes. In certainembodiments, the biomimetic organ model, or portions thereof, can bereusable, and in some embodiments, the biomimetic organ model, orportions thereof, can be disposable.

The following examples are offered to more fully illustrate theinvention, but are not to be construed as limiting the scope thereof.

EXAMPLES Example 1 Five-Layer Organ Model

The first and second channel slabs and the chamber slab of the modelwere formed using soft lithography techniques, in which the PDMS mixturewas poured over the mold, and the slabs were allowed to cure. Themicrochannels were etched into each of the channel slabs, with thedimensions of 10 mm×1 mm×0.15 mm (length×width×height). The chamber wasetched into the chamber slab, with the dimensions of 6 mm×3 mm×1 mm(length×width×height). See FIG. 9 for a picture of the five-layer modeland FIG. 2A-2B for a schematic of the five-layer model.

In order to test whether the cells in the gel layer can be fed via thechannels, experiments were conducted with only cells in the gel layer(example shown in FIG. 10). In particular, human lung fibroblasts andTHP-1 cells, a human macrophage-like cell line, were included in the gellayer. The gel was created by adding collagen to physiological aqueousbuffer. Additionally, or alternatively, any process yielding acollagen-based hydrogel may be used. The collagen solution to be used ingel preparation can typically be in, but is not limited to, theconcentration range of 0.1 to 10 mg/ml of collagen. Human lungfibroblasts (100 K cells/ml) and THP-1 macrophage (50 K cells/ml) cellswere added to the gel during the liquid phase (e.g., collagen solutionat 4° C.). The side of the membranes facing the chamber slab (e.g. 242and 251) were treated with sulfo-sanpah to promote collagen/ECManchorage. The lower channel slab, lower membrane, and chamber slab werestacked. The gel was then pipetted into the chamber (e.g., 231). Afterthe upper membrane and upper channel slab was placed on top, thebiomimetic organ model was clamped and the biomimetic organ model wasplaced in the incubator at 37° C. A picture of the clamp apparatus isshown in FIG. 9. The biomimetic organ model was incubated for five days.For continuous perfusion of culture medium at 200 μL/hr in each channel,FGM-2 can be used as the medium having a reduced serum (e.g., between0-2% and 2%). The stromal cells in the gel layer of the five-layer modelexhibited greater than 99% viability (FIG. 10). Thus, it wasdemonstrated that the cells in the gel layer can be fed via the channelsin the full five-layer assembly.

Next, the cellular physiology of the cell-lined fluidic channels withthe gel layer of the five-layer model was examined (FIG. 11). In thisinstance, the upper channel contained human lung endothelial cellscultured with commercially available medium from the supplying vendor ofthe cells. The lower channel contained small airway epithelial cellswith similar specific medium, both from same vendor. The gel was createdby adding collagen to physiological aqueous buffer at a concentration of2 mg/ml and kept at 4° C. Human lung fibroblasts (100 K cells/ml) wereadded to the gel during the liquid phase. The side of the membranesfacing the chamber slab were treated with sulfo-sanpah to promotecollagen/ECM anchorage. The lower channel slab, lower membrane, andchamber slab were stacked. The gel was then pipetted into the chamber.After the upper membrane and upper channel slab was placed on top, thebiomimetic organ model was clamped. The endothelial and epithelial cellswere then introduced via injection into the channel after presoakingwith medium and ECM coating. FIG. 11 is a phase contrast image, takingduring the culture period, depicting the interface between the gel andtwo membranes. The culture period, in this example, was 1 week. However,the culture period can have a longer duration (e.g., several weeks).

Example 2 Five-Layer Lung Fibrosis Model

This example presents a microengineered modular platform that leveragesthree-dimensional cell culture in a compartmentalized microdevice toreplicate organ-specific alterations in the cellular composition,soluble microenvironment, tissue microarchitecture and local changes inthe mechanical properties of stromal tissue during fibrosis. This systemcombines tissue-engineered hydrogel constructs impregnated with humanfibroblasts with perfusable microchannels to mimic the stromal-vascularand stromal-epithelial interface.

The ability to tune fibrotic responses using this model was demonstratedby varying the microenvironment to form a normal stroma consisting ofquiescent human lung fibroblasts (HLFs) or to induce the development offibrotic foci comprised of proliferating HLFs and a dense ECM.Furthermore, this example demonstrated the potential of this system fortherapeutic screening by showing attenuated fibrotic responses viainhibition of integrin-mediated signaling known to promote organfibrosis in vivo.

The first and second channel slabs and the chamber slab of the model wasformed using soft lithography techniques, in which the PDMS mixture waspoured over the mold, and the slabs were allowed to cure. Themicrochannels were etched into each of the channel slabs, with thedimensions of 10 mm×1 mm×0.15 mm (length×width×height). The chamber wasetched into the chamber slab, with the dimensions of 6 mm×3 mm×1 mm(length×width×height).

In the first series of validation experiments, fibrotic responses ofstromal cells embedded in the gel layer were induced by varying theserum concentration in the culture media. Incubating the gel layercontaining the NHLF cells for 12 days in 2% serum lead to increasedcellular density indicative of fibrotic change, as indicated bylive/dead staining (FIG. 12). Incubating the gel layer containing theNHLF cells in 2% serum lead to cellular proliferation indicative of afibrotic response, as the cells are very dense relatively and the gelhas detached and begun to contract and fold over. By day 16, treatmentwith 0.2% serum lead to fibrotic changes and treatment with 2% serumlead to fibrotic stroma (FIGS. 13 and 14; stained for fibronectin (FN)and smooth muscle actin (SMA)). Changes with the 0.2% at day 16 wereminor in comparison to 2% serum but more fibrotic than 0% serum. Theseresults demonstrate the ability to visualize and measure subtlevariations in organ-specific fibrotic responses. Detachment of the gellayer from the chamber was observed in most constructs cultured with 2%serum for 16 days (FIG. 15). Fibrotic foci-like structures with densefibronectin matrix and collections of polygonal cells (indicative ofpathological myofibroblast differentiation) appeared in constructscultured with 0.2% serum for 16 days (FIG. 16; stained for FN and SMA).

When the serum concentration was reduced from 0.2% to 0%, after 28 daysthe cells were quiescent and no contraction of the gel layer occurred(FIG. 17; arrows denote the few dead cells). Here, the cells werecultured in 0.2% in a 2D culture prior to use in the 3D model to promotelower levels of proliferation and ensure a more quiescent phenotype in3D culture. The cells are normally grown in 2% serum but in thisexperiment they were cultured in 0.2% serum to slow down their rate ofgrowth. They were placed in 0% serum concentration in the model, andthey stay at 0% serum for up to 28 days as shown in figure with highviability. The live/dead staining in FIG. 17 demonstrated quiescencebased on low cell density after a long period of culture.

The presence of glioblastoma-1 (Gli-1), a marker of myofibroblast cells,present in fibrotic lesions, indicated that this is a valid fibrosismodel as activity of Gli-expressing cells is a relevant pathologicalfeature of the in vivo disease (FIG. 18) as shown in Kramman et al.,Cell Stem Cell. 16(1):51-66 (2015). In the gold standard model mousebleomycin model depicted in FIG. 18, the staining pattern observed inthe mouse model of lung fibrosis is similar to what is observed in thedisclosed engineered human model.

Using different serum concentrations demonstrated the ability to measureincreased in fibrotic outputs including cell proliferation,extracellular matrix ECM production, and changes in stromal cell shapein areas of intense ECM production. The 3D nature of the cell cultureand preservation of stromal tissue geometry (e.g. preventing contractionand detachment) was important to modeling fibrosis.

Example 3 Five-Layer Injury Model

In this study, the development of the organ injury model was examined. Abiomimetic lung model was fabricated as indicated in Example 1.

The serum concentration studies above in Example 2 is one example oftunable fibrosis in the model. In this example, we studied an agentinduced injury model. Injured epithelial cells release sonic hedgehog(SHH), so SHH was added exogenously to determine if a fibrotic responsecan be induced. The initial conditions (e.g., cell density, gelconcentration, etc.) did not change from the previous examples (Examples1 and 2). However, the agent used is different in Example 3 fromExamples 1 and 2. For example, SHH was added at 500 ng/ml to produce thepro-fibrotic effect.

As demonstrated in FIG. 19, a fibrotic response can be induced bytreating the cells with SHH.

Example 4 Modulation of Fibrotic Disease Processes Using the BiomimeticFive-Layer Lung Fibrosis Model

This example examined the regulation of the fibrotic pathway usinginhibitors to reduce serum-induced fibrosis. In order to investigatethis, PP2 and separately retinoic acid (RA) were added to the cellculture media. PP2 is a non-selective proto-oncogene tyrosine-proteinkinase Src (SRC kinase) inhibitor. Src kinases transduce signals thatcontrol normal cellular processes such as cell proliferation, adhesionand motility. PP2 is known to promote a deactivated/quiescent state ofcultured (myo)fibroblasts by inhibiting activation pathways. Thesekinases are found on integrin signaling complexes and have been shown toregulate integrin signals. Therefore, blocking SRC kinases effectivelyblocks integrin signaling intracelluarly without directly interferingwith cell adhesion. Retinoic acid is involved in extracellular matrixbiosynthesis.

A biomimetic lung model was fabricated as indicated in Example 1.

The cells for the PP2 study were cultured in 2D with 2% serum and thenswitched to 0.2% in the model. 2 μM of PP2 was added to the medium 24hours after assembly of the model and maintained for the duration of thestudy. PP2 reduced fibrosis, demonstrating this could be used as ascreening platform for inhibitors of fibrosis (FIG. 20). The initialconditions (e.g., cell density, gel concentration, etc.) did not changefrom the previous examples (Examples 1, 2, and 3). However, the agentused is different in Example 3 from Examples 1 and 2.

For retinoic acid treatment, the cells and densities were the same asindicated for FIG. 11 of Example 1. The cells were cultured in 0.2 or 2%serum with or without 2 μM RA (0.2% serum) or 10 μM RA (2% serum),following similar steps as the previous examples (e.g., Examples 1-3).FIG. 21 depicts the RA inhibited serum-induced fibrotic response.

Example 5 Modeling Injury Induced Fibrosis Using the BiomimeticFive-Layer Lung Fibrosis Model

Typically organ fibrosis occurs secondarily to an organ injury. Acritical aspect of modeling related inflammatory and fibrotic diseaseprocesses absent in current state-of-the-art models of tissue fibrosisis the incorporation of resident immune cells, such as macrophages,which are present in the stroma at the location of organ injury and playa key role in mediating the organ injury response, which whenpathologically altered entails fibrotic progression. Gel anchorage andincorporation of resident immune cells are differentiatingcharacteristics compared to other platforms such as the so-called “Scarin a jar” platform. The biomimetic five-layer lung fibrosis model wasused to examine injury induced fibrosis, including macrophagedifferentiation.

A biomimetic lung model was fabricated as indicated in Example 1, andthe cells were plated and cultured as indicated for FIG. 11. Primarymonocytes derived from healthy human donors were used instead of THP-1cells (e.g., a cell line derived from leukemia). As illustrated by thisexperiment, engineered stromal microenvironment can permit and/orpromote differentiation of human blood monocytes into tissue macrophagecells.

In the absence of NHLF, monocytes did not proliferate and were notviable (FIG. 22). After the addition of NHLF and culturing for 7 dayswithout serum, the cells began to differentiate and express CD11b (FIG.22), which is an integrin complex that the cells use to adhere andmigrate through the tissue. Under the same conditions, the cells alsobegan to differentiate and express CD206 (FIG. 22), which is a marker ofdifferentiated tissue macrophages.

M2 is a phenotype of tissue macrophages, and can be further elevated byIL-4 and produce high levels of IL-10, TGF-beta and low levels of IL-12.M2 macrophages are known to decrease inflammation, and would be presentpost tissue injury. Culturing the cells in the presence of M2-polarizedmacrophages promoted fibrosis in the microengineered stromal tissue gellayer, while M1-polarized macrophages did not (FIG. 23). Culturing thecells for 13 days in the M2 conditioned media (contains the naturalmixture of factors secreted by M2 macrophases cells) induced thepresence of Gli-1 marker of myofibroblast cells (FIG. 24). The arrowindicates a cluster of cells that co-express SMA and Gli-1 at highlevels. These would be the cells that are found in fibrotic foci in vivoand serves as a validation of the model compared to what is known fromorgan fibrosis models in mice.

Example 6 Biomimetic Five-Layer Liver Fibrosis Model

The first and second channel slabs and the chamber slab of the model wasformed using soft lithography techniques, in which the PDMS mixture waspoured over the mold, and the slabs were allowed to cure. Themicrochannels were etched into each of the channel slabs, with thedimensions of 10 mm×1 mm×0.15 mm (length×width×height). The chamber wasetched into the chamber slab, with the dimensions of 6 mm×3 mm×1 mm(length×width×height).

To realize a liver model, primary human lung fibroblasts were replacedwith primary human hepatic stellate cells. The liver-specific stromalcells implicated in hepatic fibrosis. HHSC were acquired from Promocelland maintained in a vendor-supplied culture medium, although anystandard fibroblast medium such as FGM-2 with low serum concenrationscan also be used as a suitable substitute. To realize furtherorgan-specific embodiments, stromal cells derived from the organ ofchoice can form the basis of any particular organ-specific fibrosismodel.

Increased levels of serum also induced fibrosis in the liver model (FIG.24).

The present disclosure is well adapted to attain the ends and advantagesmentioned as well as those that are inherent therein. The particularembodiments disclosed above are illustrative only, as the presentdisclosure can be modified and practiced in different but equivalentmanners apparent to those skilled in the art having the benefit of theteachings herein. Furthermore, no limitations are intended to thedetails of construction or design herein shown, other than as describedin the claims below. It is therefore evident that the particularillustrative embodiments disclosed above can be altered or modified andall such variations are considered within the scope and spirit of thepresent disclosure. Various publications, patents and patent applicationare cited herein, the contents of which are hereby incorporated byreference in their entireties.

1. A multi-layer biomimetic organ model comprising: a body, having atleast two channel slabs and at least one chamber slab; at least onemicrochannel disposed in each of the at least two channel slabs; atleast one chamber disclosed in the at least one chamber slab; at leastone membrane disposed between at least one channel slab and at least onechamber slab, the membrane having a first side facing at least onemicrochannel and a second side facing at least one chamber; a gel layerdisposed in the at least one chamber; and at least one device to deliverat least one agent through at least one of the microchannels, wherein alayer of cells comprising epithelial cells are coating at least thefirst side of the first membrane.
 2. A multi-layer biomimetic organmodel comprising: a body, having a first and second channel slab and achamber slab disposed therein; a microchannel disposed in each of thefirst and second channel slab; a chamber disclosed in the chamber slab;a first membrane disposed between the first channel slab, the membranehaving a first side facing the first microchannel and a second sidefacing the chamber; a second membrane disposed between the secondchannel slab, the membrane having a first side facing the chamber and asecond side facing the second microchannel; a gel layer disposed in thechamber; and at least one device to deliver at least one agent throughat least one of the microchannels, wherein a layer of cells comprisingepithelial cells are coating at least the first side of the firstmembrane and a layer of cells comprising endothelial cells are coatingthe second side of the second membrane.
 3. The multi-layer biomimeticorgan model of claim 1, wherein the channel slabs and chamber slab areindividually comprise glass, metal, alloy, plastic, wood, paper, or apolymer.
 4. The multi-layer biomimetic organ model of claim 1, whereinthe membrane comprises polyester thin clear fabric,polydimethylsiloxane, polymeric compounds, or natural membranes, whereinthe natural membrane comprise collagen, laminin, or a combinationthereof.
 5. The multi-layer biomimetic organ model of claim 1, whereinthe first microchannel, second microchannel, or both has a width fromabout 0.01 nm to about 1 cm and a length from about 1 mm to about 10 mm.6. The multi-layer biomimetic organ of claim 1, wherein the epithelialcells comprise pulmonary, hepatic, or renal epithelial cells and theendothelial cells comprise endothelial cells from the same organ.
 7. Themulti-layer biomimetic organ model of claim 1, wherein at least onelayer of cells further comprises at least one type of macrophages,dendritic cells, microbial cells or mixtures thereof.
 8. The multi-layerbiomimetic organ model of claim 1, wherein the gel layer is embeddedwith tissue or cells.
 9. The multi-layer biomimetic organ model of claim1, wherein the gel layer comprises extracellular matrix proteins,wherein the extracellular matrix proteins are selected from the groupconsisting of, collagen, fibronectin, laminin, hyaluaronic acid, andmixtures thereof.
 10. The multi-layer biomimetic organ model of claim 9,wherein the tissue or cells comprise basal stromal tissue or cells. 11.The multi-layer biomimetic organ model of claim 1, wherein the gel layerfurther comprises at least one type of macrophages, dendritic cells,microbial cells or mixtures thereof.
 12. The multi-layer biomimeticorgan model of claim 1, wherein the epithelial cells are obtained from ahealthy human organ or a chronically diseased human organ.
 13. Themulti-layer biomimetic organ model of claim 1, for use in identifyingpharmaceutical compositions that treats or prevents an organ disease.14. The multi-layer biomimetic organ model of claim 1, for use inidentifying agents harmful to the organ.
 15. A method for fabricating amulti-layer biomimetic organ model comprising: (a) fabricating a body,the body having at least two channel slabs and at least one chamber slabdisposed thereon; (b) etching a microchannel disposed on each of the atleast one channel slabs; (c) etching a chamber disposed on the at leastone chamber slab; (d) inserting a cell embedded gel layer in the atleast one chamber; (e) inserting a first membrane between a firstmicrochannel and a chamber, the membrane having a first side facing thefirst microchannel and second side facing the chamber; (f) inserting asecond membrane between a second microchannel and the chamber, thesecond membrane having a first side facing the chamber and second sidefacing the second microchannel; (g) adhering epithelial cells on asurface of at least the first side of the first membrane; (h) couplingat least one device to deliver an agent; and (i) delivering an agent toat least one microchannel.
 16. A method for fabricating a five-layerbiomimetic organ model comprising: (a) fabricating a body, the bodyhaving a first and second channel slab and a chamber slab disposedthereon; (b) etching a microchannel disposed on each of the first andsecond channel slab; (c) etching a chamber disposed on the chamber slab;(d) inserting a cell embedded gel layer in the chamber; (e) inserting afirst membrane between the first microchannel and the chamber, themembrane having a first side facing the first microchannel and secondside facing the chamber; (f) optionally inserting a second membranebetween the second microchannel and the chamber, the membrane having afirst side facing the chamber and second side facing the secondmicrochannel; (g) adhering epithelial cells on a surface of at least thefirst side of the first membrane; (h) coupling at least one device todeliver an agent; and (i) delivering an agent to at least onemicrochannel.
 17. The method of claim 15, wherein adhering epithelialcells comprises seeding a high density cell suspension upon the firstside of the membrane and incubating the cells suspension for about 2 toabout 4 hours.
 18. The method of claim 15, further comprising casting agel embedded with connective tissue and attaching the gel to thechamber.
 19. The method of claim 18, wherein casting a gel comprisespipetting the gel with or without cells within the chamber and allowingthe gel to solidify.
 20. The method of claim 15, further comprisingintegrating macrophages, dendritic cells, and/or microbial cells amongat least one layer of cells.